Nucleic acid ligands and uses therefor

ABSTRACT

The present invention relates to novel nucleic acid molecules or ligands or aptamers with affinities for specific target molecules, and uses of such molecules. The target molecules are fibrillar proteins in all forms of the protein that is to say its monomeric, pre-fibrillar, protofibrillar and mature fibrillar forms. The molecules of the present invention are therefore useful as diagnostic or therapeutic or screening agents, or as potential lead compounds for ralionalised drug design.

The present invention relates to novel nucleic acid molecules or ligandswith affinities for specific target molecules, and uses of suchmolecules, especially but not particularly as diagnostic or therapeuticor screening agents, or as potential lead compounds for rationaliseddrug design.

BACKGROUND TO THE INVENTION

A number of proteins undergo specific aggregation into amyloid fibrilsin vivo leading to a variety of pathological disorders, collectivelyknown as amyloidoses¹. These diseases are characterised by thedeposition of normally soluble proteins or peptides into insolublefibrillar arrays. About 20 different proteins have been identified todate as the fibrillar component in different human amyloid diseases. Insome cases, the intact wild-type protein is involved (β2-microglobulin,(β2m), calcitonin, insulin and amylin), whilst in other cases (amyloidprecursor protein (APP), lysozyme, transtyretin, gelsolin and others)mutated forms of the protein or protein fragments are involved. However,it is not known how normally soluble, proteins or peptides transforminto the ordered array of β-strands typical of amyloid, although partialdenaturation of the native protein or folding of more highly denaturedstates is thought to be a critical first step^(2,3). Equallyimportantly, the specie(s) responsible for the toxicity of amyloidis/are also unknown. There is some evidence to suggest that earlyoligomers or other fibril assembly intermediates, rather than the fullyassembled amyloid fibrils could be the toxic agent⁴⁻⁷.

In recent studies, it has been shown that amyloid-like fibrils can begenerated in vitro from proteins not known to be associated withdisease⁸, suggesting that this structure could be the ultimate groundstate of all polypeptide chains. In accord with this view, the sequencesof proteins and peptides known to be involved in human amyloid diseasesalso show no similarity in sequence or overall properties, for examplesecondary structure propensity and hydrophobicity. In all cases, theprotein fibrils formed have a common overall architecture in whichβ-strands orient perpendicular to the fibre long axis into an arrayknown as a cross-β structure. Fibrils are typically long, unbranched,approximately 10 nm in diameter and are formed from simpler units knownas protofilaments or filaments. Amyloid fibrils thus give rise to adistinct X-ray fibre diffraction pattern, and are identified by theirunique ability to bind the dyes Congo red and Thioflavin T (Thio-T),resulting in characteristic spectral changes. However, fibril formationby peptides and proteins requires specialised equipment, whilst thestates of aggregation of pre-fibrillar forms of these species aredifficult to detect by current methods. Therefore imaging of fibrillarspecies in tissue samples has hitherto been difficult and largelyrestricted to end stage species. There is therefore a need for simple,routine screening protocols of biological tissues for all aggregated andfibrillar species.

Examples of amyloidosis disease include new variant CJD, mature onsetdiabetes, Alzheimer's disease and dialysis-related amyloidosis (DRA).Alzheimer's disease is characterised by large plaques in the brain thatcontain necrotic neurons, neurofibrillary tangles containing the tauprotein and fibrils composed of peptides derived from the Alzheiner'sPrecursor Protein (APP)⁴. With regard to the treatment of Alzheimer'sdisease, studies with monoclonal antibodies directed against Aβ1-40 haveshown that neurodegeneration can be halted and/or reversed in animalmodels, suggesting that vaccination against Aβ1-40 could be a successfultherapy⁹. Unfortunately despite the promising initial animal work,recent clinical trials of this therapy failed due to cross-reactivitywith the APP precursor protein leading to inflammation of the brain.Accordingly there is still no effective therapy for this disease.

Dialysis-related amyloidosis (DRA) involves the human protein, β₂m, theaggregation of which into amyloid fibrils is the cause of the disorderdialysis-related amyloidosis ¹⁰. This protein forms the non-covalentlybound light chain of the class I MHC complex. As part of its normalcatabolic cycle, β₂m dissociates from the heavy chain, whereupon it iscarried in the serum to the kidneys where it is degraded and excreted.As a consequence of renal failure therefore, the concentration ofcirculating β₂m increases, and, by a mechanism currently unknown, thefull-length wild-type protein aggregates to form amyloid fibrils thattypically accumulate in the musculo-skeletal system. DRA is a common andserious complication of long-term haemodialysis, currently affectingmore than 750,000 people world-wide and serious morbidity develops inmore than 90% of patients undergoing dialysis for a period of 10 or moreyears. Despite the identification of β₂m as the culprit protein in DRAmore than 16 years ago¹¹, there are currently no therapies for thedisease other than organ transplantation.

Accordingly, determining the structure of amyloid fibrils, together withdiscovery of agents able to discriminate between the differing forms ofamyloid aggregates, and being able to regulate or alter theiraggregation properties would offer an immediate advantage for the designof therapeutic and diagnostic agents against amyloidosis. In addition,specific agents directed against either soluble Alzheimer's Aβ1-40peptide or β₂m, or their proto- or fibrillar forms, that could preventamyloid formation and hence halt progression, and even reverse,neuro-degeneration due to plaque deposition or β₂m amyloid build upwould be of immediate therapeutic value of benefit to the pharmaceuticalindustry and sufferers of the diseases. Furthermore, if the 20amyloidosis diseases have a common underlying molecular mechanismtherapeutic reagents created against one type of amyloid could be usefulagainst many others.

Novel synthetic DNA/RNA ligands, known as aptamers, have been defined¹²as artificial nucleic acid ligands that can be generated against aminoacids, drugs, proteins and other molecules. They are isolated fromcomplex libraries of synthetic nucleic acids by an iterative process ofadsorption, recovery and reamplification.

RNA aptamers are nucleic acid molecules with affinities for specifictarget molecules. They have been likened to nucleic acid antibodiesbecause of their ligand binding properties. They may be considered asadvantageous agents over antibodies for a variety of reasons.Specifically, they are soluble in a wide variety of solution conditionsand concentrations and their binding specificities are largelyundisturbed by reagents that have significant effects on antibodyreagents, e.g. detergents and other mild denaturants, moreover they arerelatively cheap to isolate and produce compared to antibodies. They mayalso readily be modified to generate species with improved propertieswhereas antibodies cannot always be adapted easily. Extensive studiesshow that nucleic acids are largely non-toxic and non-immunogenic andaptamers have already found clinical application¹³, whereas antibodiesbeing proteins are strongly immunogenic and require extensive andexpensive manipulation to be used in humans.

However, a serious disadvantage associated with RNA aptamers is thatnatural RNAs are unstable in biological fluids. It is known in the priorart how to improve stability by chemically modifying RNAs so as to blocknuclease action at 5′ and 3′ ends, and throughout the length of themolecule. However, such chemical modification can ultimatelydetrimentally alter the binding properties of the RNA and hence renderthem ineffective.

It is known from the prior art how to isolate aptamers from degeneratesequence pools by repeated cycles of binding, sieving and amplification.Such methods are described in U.S. Pat. No. 5,475,096, U.S. Pat. No.5,270,163 and EP0533 38 and typically are referred to as SELEX(Systematic Evolution of Ligands by EX-ponential Enrichment). The basicSELEX system has been modified for example by using Photo-SELEX whereaptamers contain photo-reactive groups capable of binding and/or photocross-linking to and/or photo-activating or inactivating a targetmolecule. Other modifications include Chimeric-SELEX, Blended-Selex,Counter-SELEX, Solution-SELEX, Chemi-SELEX, Tissue-SELEX andTranscription-free SELEX which describes a method for ligating randomfragments of RNA bound to a DNA template to form the oligionucleotidelibrary. However, these methods even though producing enrichedligand-binding nucleic acid products, still produce unstable products.In order to overcome the problem of stability it is known to createenantiomeric “spiegelmers” (WO 01/92566). The process involves initiallycreating a chemical mirror image of the target, then selecting aptamersto this mirror image and finally creating a chemical mirror image of theSELEX selected aptamer. By selecting natural RNAs, based on D-ribosesugar units, against the non-natural enantiomer of the eventual targetmolecule, for example a peptide made of D-amino acids, a spiegelmerdirected against the natural L-amino acid target can be created. Oncetight binding aptamers to this target are isolated and sequenced, theLaws of Molecular Symmetry mean that RNAs synthesised chemically basedon L-ribose sugars will bind the natural target, that is to say themirror image of the selection target. This process is convenientlyreferred to as reflection-selection or mirror selection and the L-ribosespecies produced are significantly more stable in biologicalenvironments, are less susceptible to normal enzymatic cleavage and arenuclease resistant.

STATEMENT OF THE INVENTION

According to a first aspect of the invention there is provided apurified and isolated non-naturally occurring nucleic acid ligand to afibrillar protein, wherein said ligand is an RNA ligand selected fromthe group comprising:

-   -   (i) the nucleic acids depicted in any one of SEQ ID NOS: 1-55 or        58-105;    -   (ii) having the corresponding DNA or RNA sequences of any one of        SEQ ID NOS: 1-55 or 58-105, or the corresponding fully        complementary sequences thereof or their L-ribose derivatives;    -   (iii) derivatives of the sequence depicted in any one of SEQ ID        NOS: 1-55 or 58-105 having at least about 60%, 70%, 80% or 90%,        sequence identity to any one of the nucleotide sequences, and        which have a binding affinity to a fibrillar protein.

Accordingly the nucleic acids of the present invention may be RNAs ortheir L-ribose derivatives or may be the DNAs encoding the RNAs or theirL-ribose derivatives.

Reference herein to fibrillar protein is intended to include all formsof the protein that is to say its monomeric, pre-fibrillar,protofibrillar and mature fibrillar forms.

Sequence identity is the similarity between two nucleic acid sequences,or two amino acid sequences, and is expressed in terms of the percentagesimilarity between the sequences. The higher the percentage, the moresimilar the two sequences are. Homologues or orthologues of the protein,and the corresponding cDNA or gene sequence, will possess a relativelyhigh degree of sequence identity when aligned using standard methods.This homology will be more significant when the orthologous proteins orgenes or cDNAs are derived from species that are more closely related(e.g., human and chimpanzee sequences), compared to species moredistantly related (e.g. human and C. elegaus sequences).

Reference herein to an aptamer is intended to include nucleic acidmolecules with binding affinities for specific target molecules,especially but not exclusively RNA nucleic acid molecules.

Preferably, the nucleic acid ligand or aptamer is substantiallyhomologous to and has substantially the same ability to bind saidfibrillar protein as a ligand selected from the group comprising thenucleic acids depicted in any one of SEQ ID NOS: 1-55 or 58-105.

Preferably, the nucleic acid ligand or aptamer has substantially thesame structure and the same ability to bind said fibrillar protein as aligand selected from the group comprising the nucleic acids depicted inany one of SEQ ID NOS: 1-55 or 58-105.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated integer or group of integers but not the exclusion of anyother integer or group of integers.

Reference herein to binding affinity is intended to include bindingaffinities expressed as equilibrium dissociation constants, Kd, in thefrom sub-millimolar to picomolar.

In one embodiment of the invention, we have investigated aptamersselected against β2m, Aβ1-40 and Aβ1-42, as both their L- and D-aminoacid molecules, and their fibrillar forms. The monomeric targets beingconveniently referred to as T1, the proto- and more mature fibrils as T2and T3, respectively for the Aβ targets. The equivalent β2m targetsbeing designated monomeric, “curly” fibrils and mature “rod-like”fibrils. Our initial aim was to better understand the molecular basis ofsequence-specific recognition of RNAs by proteins and peptides. In thecourse of this work, we have not only isolated tight binding RNAaptamers against the targets but surprisingly and unexpectedly, theaptamers discovered have properties that make them potentially usefulentities. This is in contrast to the species reported previously thatbind amyloid plaques¹⁴.

Accordingly, one aspect of the invention pertains to isolated nucleicacid molecules (e.g., RNAs) comprising a nucleotide sequence which has abinding affinity for a fibrillar protein or biologically active portionsthereof, as well as nucleic acid fragments suitable as primers orhybridization probes for the detection or amplification of nucleic acidligands for fibrillar proteins. In particularly preferred embodiments,the isolated nucleic acid molecule comprises one of the nucleotidesequences set forth in SEQ ID NO: 1-55 or 58-105 or a complement thereofof one of these nucleotide sequences. In other particularly preferredembodiments, the isolated nucleic acid molecule of the inventioncomprises a nucleotide sequence which hybridizes to or shows at leastabout 50%, preferably at least about 60%, more preferably at least about70%, 80% or 90%, and even more preferably at least about 95%, 96%, 97%,98%, 99% or more homologous to a nucleotide sequence as in SEQ ID NOS:1-55 or 58-105, or a portion thereof.

Preferably, the aptamers of SEQ ID NOS: 1 to 16 have a preferentialbinding affinity to a D-amino acid Aβ1-40 monomeric target.

Preferably, the aptamers of SEQ ID NOS: 17 to 36 have a binding affinityto a D-amino acid β1-40 pre-fibrillar target.

Preferably, the aptamers of SEQ ED NOS: 37 to 55 have a binding affinityto a D amino acid Aβ1-40 protofibril target.

Preferably, the aptamers of SEQ ID NOS: 58 to 71 have a binding affinityto native β2-microglobulin protein target. Preferably, the aptamers ofSEQ ID NOS: 72 to 90 have a binding affinity to a β2-microglobulinimmature fibril protein target.

Preferably, the aptamers of SEQ ID NOS: 91 to 105 have a bindingaffinity to a β2-microglobulin mature fibrillar protein target.

Preferably, the aptamers of the present invention further include anyone or more of the following features as herein recited such as afluorescent label, an imaging label or a flanking region.

The core for the aptamer is the random RNA oligonucleotide sequence,which is flanked by a 5′ and 3′ constant sequence (SEQ ID NOS:56, 57,106 and 107) that provide primer hybridisation sites for Klenowextension, cDNA synthesis, polymerase chain reaction amplification andT7 RNA polymerase tanscritption, all of which are involved in the SELEXprotocol. It should be appreciated that the selection of the constantflanking region is important to ensure optimum efficacy of SELEX. The 3′flanking region acts as the attachment site for MMLV reversetranscriptase primer that converts the RNA aptamers to DNA. The 5′flanking sequence acts as the point of attachment for PCR primers whichinitiate amplification of the selected sequence.

According to a yet further aspect of the invention there is provided avector comprising or encompassing at least one or more aptamer of thepresent invention.

This aspect of the invention pertains to vectors, preferably expressionvectors, containing a nucleic acid having a binding affinity for afibrillar protein (or a portion thereof). As used herein, the term“vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. One type of vector isa “plasmid”, which refers to a circular double stranded DNA loop intowhich additional DNA segments can be ligated. Another type of vector isa viral vector, wherein additional DNA segments can be ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmild” and “vector” can be used interchangeably asthe plasmid is the most commonly used form of vector. However, theinvention is intended to include such other forms of expression vectors,such as viral vectors (e.g., replication defective retroviruses,adenoviruses and adeno-associated viruses), which serve equivalentfunctions.

According to a yet further aspect of the invention there is provided ahost cell including at least one aptamer or a vector comprising at leastone aptamer of the present invention.

According to a yet further aspect of the invention there is provided anaptamer binding motif comprising a peptide sequence derived from humanβ2m that retains the ability of the whole protein to form amyloidfibrils. This sequence, in either the natural L-amino acid or un-naturalD-amino acid sequence, is NH₂-FYLLYYTE-COOH (SEQ ID NO: 111) orNH₂-DWSFYLLYYTEFT-COOH (SEQ ID NO:112) or NH₂-DWSFYLLYYTEFTPTEKDEYA-COOH(SEQ ID NO:113), where the designations NH₂ and —COOH show the chemicalconnectivity of the peptides and the sequence in between represents thestandard one-letter code for the amino acids¹⁵. Note, in each case theamino terminus of these peptides can by either free or acetylated, andlikewise the carboxy terminus can be either free or in the form of anamide.

In the course of investigating the aptamers of the present invention wehave identified two fibrillar forms of the protein, an intermediate,globular (immature) form and a mature fibrillar form. Accordingly thisnew form represents a target for identifying/developing aptamersdirected thereto and may be of value in identifying father agents thatcan control/prevent/reverse fibril formation.

According to a yet further aspect of the invention there is providedaptamers directed against the binding motif as herein before described.

According to a yet further aspect of the invention there is provided anaptamer directed against the cross β-core structure.

There is a common secondary structure in amyloids in that whatever thefold of the soluble form they share a secondary structure core composedof a β-strand orientated perpendicular to the fibril long axis. Thus, itis envisaged that aptamers directed against this target would find useas therapeutic agents in a variety of diverse amyloid diseases.

Currently twenty human proteins are known that cause disease viamis-folding and amyloid formation. It is estimated that amyloidformation is implicated in 1:1000 deaths in the UK and could be a majorcause of tissue and organ ageing. Despite the variety of amyloid formingproteins the amyloid structure itself has a common secondary structuralelement kmown as a cross-β-fold that appears independent of theprotein/peptide sequence involved, although this does alter the detailedmorphology of the mature fibrils observed. Work with antibodies hasidentified common epitopes (recognition features) shared by many amyloidstructures. We therefore believe that there is/are a common apatope(s)(aptamer recognition features) which will allow anti-amyloid aptamers toact as a generic “magic bullet”. It is believed that the anti-amyloidaptamers of the present invention have the potential to cross-react withall forms of amyloid whatever its source, thus dramatically extendingtheir utility in imaging/screening and therapeutics.

According to a yet further aspect of the invention, there is provided apharmaceutical composition comprising at least one aptamer of thepresent invention.

In another embodiment of the invention the pharmaceutical compositionmay comprise a number of aptamers each with binding affinities for thesame or different forms of a fibrillar protein.

Preferably, the pharmaceutical composition includes a suitableexcipient, diluent or carrier.

According to a yet further aspect of the invention there is provided useof an aptamer of the present invention for the manufacture of amedicament for treating amyloid diseases.

It is envisaged that pharmaceuticals and medicaments utilising theaptamers of the present invention may be used to treat Alzheimer's andDRA disease conditions.

According to a yet further aspect of the invention there is provided amethod of treating a patient suffering from Alzheimer's disease or adisease associated with amyloid formation comprising administering atherapeutically effective amount of an aptamer or pharmaceuticalcomposition comprising an aptamer of the present invention.

There is evidence that βA1-40 and Aβ1-42 fibril formation is naturallyprevented by other systems that recognise the peptide and clear it fromthe blood stream. These systems seem to work less well in Alzheimer'spatients. If self-aggregation can be slowed and/or prevented and thegrowing fibrils remain soluble, it should be possible for these naturalsystems to keep up with peptide clearance.

Kidney dialysis patients suffer from induction of fibril formation intheir β2m, leading to painful deposition in joints. It is envisaged thatthe aptamers and pharmaceutical compositions of the present inventionwill be of use in treating such conditions.

Preferably, the aptamer or pharmaceutical composition comprising anaptamer is administered directly to an amyloid site or it may beadministered by an intra-venous, intra-muscular, intra-peritoneal routeand preferably may be administered on more than one occasion. Inaddition, aptamers could be administered to the blood stream, the siteof the raised level of soluble β2m, in order to stablise the solubleform of the protein.

It will be appreciated that the aptamers against the Aβ1-40, Aβ1-42 andβ2m species may be modified with fluorescent labels by simple inclusionof, for example, fluorescein-labelled UTP in in vitro tanscriptionreactions. The fluorescence properties of these molecules are sensitiveto their bound state and may be the basis of simple diagnostic screeningand imaging reagents. For instance, the state of disease progression maybe judged by staining/screening with differently labelled aptamersdirected against monomer, pre-fibril or fibrillar species and thereforepreferably the aptamers and pharmaceutical compositions comprising oneor more aptamers will have utility not only in treating diseaseconditions and ameliorating symptoms but in assaying disease preventionand progression.

According to a yet further aspect of the invention there is use of theaptamers of the present invention as a diagnostic agent for detectingthe presence and/or progression of an amyloid disease.

Aptamers of the present invention directed against the fibrillar formsof β2m and the reflection-selection aptamers against the Aβ1-40 speciesor Aβ1-42 species may preferably be suitably modified with at least onefluorescent label to allow diagnostic screening and/or at least oneimaging reagent for these conditions. For instance, the state of diseaseprogression may be monitored by staining/screening with differentlylabelled aptamers directed against individual monomer, pre-fibril orfibrillar species or a mixture of species over period of time.

According to a yet further aspect of the invention there is provided amethod of monitoring the presence and/or progression of an amyloiddisease comprising administering to a patient at least one aptamer or apharmaceutical composition or aptamer product comprising an aptameraccording to the present invention and imaging the presence of bindingof said aptamer to an amyloid fibril and optionally repeating theprocess at a later date to assess presence or progression of a diseasestate.

According to one aspect of the invention there is provided a method forthe isolation of nucleic acid ligands to a fibrillar protein targetcomprising:

-   -   (i) preparing a candidate mixture of nucleic acids;    -   (ii) contacting the candidate mixture of nucleic acids with a        biotinylated immobilised fibrillar protein, wherein nucleic        acids having an increased affinity to the fibrillar protein        relative to the candidate mixture may be partitioned from the        remainder of the candidate mixture;    -   (iii) partitioning the increased-affinity nucleic acids from the        remainder of the candidate mixture;    -   (iv) amplifying the increased-affinity nucleic acids to yield a        mixture of nucleic acids with relatively higher affinity and        specificity for binding to the fibrillar protein, whereby a        nucleic acid ligand of the fibrillar protein may be identified.

Preferably, the candidate mixture comprises single stranded nucleicacids and more preferably the single stranded nucleic acids compriseribonucleic acids.

Preferably, the target comprises at least one biotin molecule. The β2mtarget protein requires biotinylation in order for it to attach to asurface, for example a streptavidin bead, so as to be immobilisedhowever, biotinylation of native β32m may hinder fibril formation hencewe have performed this step on preformed fibrils. Our results have shownsurprisingly that a combination of pH shift and biotinylation on icedoes not destroy fibrils and that SELEX is advantageously improved.

Preferably, the fibrillar target is isolated and immobilised. We havefound surprisingly that, as exemplified by β2m, increasing the solutionpH>5 does not lead to dissociation of the fibrils as expected fromprevailing teachings¹⁶, provided that the sample is maintained on icefor up to 10 minutes. Accordingly, the modified method of the presentinvention allows for a biotinylated target to be immobilised by pHshift.

Preferably, the method further includes the step of modifying thenucleic acid ligands with a fluorescent label and/or an imaging reagent.A non-limiting example of a suitable fluorescent labels isfluorescein-labelled UTP, and non-liming example of imaging agents areuranyl acetate, and radioactive technetium and indium-labelled species,for both in vitro and in vivo applications.

Preferably, the method further includes the step of flanking saidaptamer with at least one further nucleic acid sequence comprising thenucleic acid as set forth in SEQ ID NO:56 and optionally the aptamer isflanked by a further nucleic acid sequence as set forth in SEQ IDNO:107. It will be appreciated that other constant flanking regions maybe used and the composition of the flanking regions is not intended tolimit the scope of the invention.

The method of the present invention advantageously allows for rapidselection and characterisation and is accomplished in vitro withoutrecourse to animal work. It will be appreciated that aptamers of thepresent invention will have commercial application in many areascurrently making use of antibodies, for example and without limitation,as diagnostic and screening tools and as therapeutic agents in a varietyof different disease conditions.

Preferably, the product may be further modified as hereinbeforedescribed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 represents a schematic diagram showing the growth of cross-βstrand structure from soluble monomeric precursors to generate amyloidfibrils via a conformational rearrangement (top and middle lines). Alsoshown are models for how aptamer reagents might inhibit fibrillogenesis,either by stabilising the monomeric form (top line, right hand side) orby directly blocking fibril growth (lower line).

FIG. 2 shows that an anti-fibril β2m aptamer (SEQ D NO 74), labelledwith fluorescein-UTP undergoes differential quenching and wavelengthshifting of fluorescence emission in the presence of differing forms ofthe β2m target.

FIG. 3A shows surface plasmon resonance binding curves (sensorgrams) ofthe anti-β2m fibril aptamer (SEQ ID NO 74) binding to: a) anunderivatised flow cell (red, the signal falls due to differences inrefractive index of the sample); b) a flow cell derivatised with nativeβ2m (magenta, no detectable binding under these conditions); c) a flowcell derivatised with β2m fibrils green, strong binding with slowdissociation) or d) a flow cell derivatised with mature β2m fibrils(blue, result as in (c)); FIG. 3B shows the equivalent experiments for anative aptamer (SEQ ID NO 61). Note, the anti-native aptamers were notcounter-selected against fibrils.

FIG. 4A shows a time course of Aβ1-40 fibril formation followed bytransmission electron microscopy (TEM) and FIG. 4B a the same timecourse in a continuous assay utilising the increasing Thio-Tfluorescence upon fibril binding.

FIGS. 5A and B show nitrocelluose filter-binding curves for the bindingof anti-Aβ1-40 T3 aptamers (SEQ ID NO:55 and 38 respectively) to theircognate target. 5B also shows the effects of adding aptamer SEQ ID NO:38to a mixture of Aβ1-40 undergoing fibril formation (left panel). Theupper TEM shows the formation of extended fibrils after 16 hours,whereas when the aptamer is added to an aliquot of the same sample after10 mins of fibril formation further fibril formation is severelyinhibited.

FIG. 6 shows reflection-selection apatmer (SEQ ID NO:38) binding to anatural enantiomer of its initial target and indicates the potential ofthis bio-stable aptamer to block fibril formation.

FIG. 7 shows mass spectrometry data for biotinylated β2m fibrils. Thedata suggest that under the conditions employed 1-3 biotin residues areintroduced per β2m monomer.

FIG. 8 shows thio-T assays of the various points of the β2m biotinderivatisation protocol and indicates clearly that fibrils are stillpresent after modification.

FIG. 9 shows the nature of the fibrillar β2m selection targets. Theimmature (short, curly) fibrils (left-hand side) and the mature fibrils(long, straight) on the right. The images (lower diagrams) are TEM andatomic force micrographs, respectively. The Thio-T binding potential ofeach fibrillar form is shown in the top panels.

FIG. 10 illustrates the effects of aptamers on β2m fibril formation invitro. -β2m in the absence of aptamers; □-β2m in the presence of ananti-immature fibril (SEQ. ID NO 74); ▪-β2m in the presence of theanti-monomer 10^(th) round aptamer pool (NR10) and as a negativecontrol, ◯-β2m in the presence of the naïve starting pool for theaptamers.

FIG. 11 shows the structure of human β2m and the location of E strandtarget peptides.

FIG. 12 shows the amino acid sequence of the peptides of β₂m.

MATERIALS AND METHODS

Aβ1-40 and Aβ1-42 Peptides

Fibril Formation

In order to characterise amyloid fibril formation from Aβ(1-40) usingfluorescence assays and electron microscopy, thereby isolating andimmobilising meta-stable targets for SELEX, the following method wasemployed. It will be appreciated that the method is also applicable foramyloid fibril formation from Aβ(1-42) target.

0.5 mgs of unbiotinylated Aβ(1-40) (obtained from Biosource) wasdissolved in 1 mL 99.8% trifluoroacetic acid (TFA). The TFA was thenremoved by passing a jet of filtered, dry nitrogen gas over the sample.The dried Aβ was then dissolved in 1 mL hexafluoroisopropanol (HFIP,from Fluka) so as to remove any residual TFA. The HFIP was thensimilarly removed. The Aβ was then vacuum pump dried for 1 hr to removeany residual HFIP. The resulting disaggregated peptide was stored at−20° C. until required.

10:1 ‘mixed’ (unbiotinylated+biotinylated) Aβ fibrils were used fromstocks of both forms of pre-prepared Aβ in HFIP (as described above).Appropriate volumes of each stock were mixed to produce a ˜50 μM (10:1)mixture of unbiotinylated and biotinylated Aβ in HFIP. The mixture wasdried under N₂ gas and vacuum pump dried to remove all the HFIP. The‘mixed’ treated peptide was then dissolved in an appropriate volume ofcold 1× Binding Buffer (50 mM MOPS, pH 7.4, 50 mM NaCl, 1 mM MgCl₂) byrapid vortexing. The peptide solution was stored on ice and then quicklytransferred to a 1 mL fluorescence cuvette containing a magneticstirrer. A volume of Thio-T solution was added to a final concentrationof 5 μM. The photobleaching shield from the fluorimeter (LS50B) wasremoved and a time drive scan initiated for 7200 seconds. The settingson the fluorimeter were λ_(EX)=444 nm λ_(EM)=482 nm S_(EX)=5 nmS_(EM)=10 nm. The scan was followed and 40 μL samples removed at t=100 s(corresponding to a steady fluorescence signal), 2000 s (correspondingto a slow increase in fluorescence signal), 4800 s (corresponding to anexponentially increasing in fluorescence signal); 7200 s (correspondingto a slowing of the rate of change of the fluorescence signal) and 14400 s (corresponding to the approach of saturation by the fluorescencesignal). Each of these samples were negatively stained and prepared forexamination by electron microscopy (FIG. 4A).

The first meta-stable precursor species to fibril formation or 12(appeared oligomeric under the EM) were identified by a steadyfluorescence signal, lasting up to 2000 seconds. Subsequently, T2 wasprepared by stirring the Aβ solution for 10 minutes and then storing onice for immediate use in SELEX experiments.

The next meta-stable precursor species to be identified were theprotofibrils or T3. These remained stable for up to 30 to 40 minutesafter the formation of T1 (their presence was marked by a slow increasein fluorescence signal). The end of their existence is heralded by anexponential increase in fluorescence signal (marking the formation ofelongating, maturing fibrils). T3 was prepared by stirring the Aβsolution for up to 40 minutes at 20° C. and then storing on ice forimmediate use in SELEX experiments. T1 or monomeric Aβ(1-40) was storedin HFIP and subsequently diluted in 1× binding buffer when required.

With reference to FIG. 4A there is shown an time course of Aβ1-40 fibrilformation followed by transmission electron microscopy (TEM), as will beapparent protofilaments are observed after 30-40 minutes of theprocedure. FIG. 4B shows a corresponding time course of amyloid fibrilformation using Thio-T binding fluorescence. Our results showed thatboth L- and D-amino acid versions of Aβ1-40 exhibited the same behaviourwith respect to the kinetics and morphology of fibril formation

SELEX

In order to carry out at least 9 rounds of automated selection of RNAaptamers against three precursor meta-stable species of amyloid fibrilformation T1 (native Aβ); T2 (oligomeric Aβ); T3 (protofibrillar Aβ),the following protocol was adopted. Using the following reagents, a T7MEGA shortscript transcription kit [Ambion], RNase inhibitor[invitrogen], Upstream primer [5′ AAT TAA CCC TCA CTA AAG GGA ACT GTTGTG AGT CTC ATG TCG AA . . . 3′] (SEQ ID NO:56), Downstream primer [5′TAA TAC GAC TCA CTA TAG GGA GAC AAG ACT AGA CGC TCA A . . . 3′] (SEQ IDNO:106) and Random 50 mer [5′ AAT TAA CCC TCA CTA AAG GGA ACT GTT GTGAGT CTC ATG TCG AA-N₅₀-TT GAG CGT CTA GTC TTG TCT 3′] (SEQ ID NO:107).

Each target was pre-prepared as hereinbefore described. Targets wereimmobilised onto Magnetic STREP-Microspheres as described by themanufacturer's instructions and stored at 4° C. until required. Aprogram Amb T1-3 was written for the automated selection of RNA aptamersagainst all three targets. The program was modified from that of Cox andEllington¹⁹ in the following manner:

-   -   (i) The DNA starting pool was prepared by amplifying a random 50        mer with the use of fixed upstream and downstream primer        regions. The radioactively labelled pool was gel purified,        quantified and subsequently used to generate the first RNA pool    -   (ii) Ambion kit transcription reagents (transcription buffer,        NTPs, T7 polymerase) (Ambion) were used.    -   (iii) Mineral oil was used in the PCR step    -   (iv) Magnetic STREP-microspheres by Abgene were used instead of        Dynabeads

Ligation and Cloning of RT-PCR Products Generated after 9 Rounds ofAutomated SELEX

RT-PCR products collected at the end of Round 9 of automated SELEX wereamplified under the following PCR conditions: 10×PCR buffer, MgCl₂ (50mM) to a final concentration of 1.5 mM; Taq DNA polymerase [Invitrogen]dNTP mix [Gibco BRL] to a final concentration of 0.02 mM; Upstream (SEQID NO:56) and Downstream primers (SEQ ID NO:106); Dimethyl Sulphoxide(DMSO); QIAEX II kit for gel purification [Qiagen]; TA TOPO Cloningsystem [(Invitrogen].

Fluorescently-Labelled Aptamers

A fluorescently-labelled T3 clone RNA (aptamer against target 3) (SEQ IDNO:38) was prepared and the effect on its fluorescence signal on bindingto T3 was determined. The transcription mixture was prepared as follows:100 ng DNA template 10 μLs; fluorescein RNA labelling mix [Roche] 2 μLs;10× transcription buffer 2 μLs; RNase free water 18 μLs to 2 μLs; T7 RNApolymerase 2 μLs .

The mixture was incubated at 37° C. for 2 hrs. 2 μLs DNase I was thenadded to remove the template (incubation at 37° C. for 15 minutes).Reaction was stopped by adding 2 μLs 0.2 M EDTA, pH8. The transcript waspurified by Phenol:Chloroform extraction and desalted by passing samplethrough a G-25 column (Amersham). The purified fluorescently-labelledRNA was EtOH precipitated to remove all unincorporated NTPs and storedin 10 μLs 1× Binding buffer at −20° C. until required.

A fluorescence emission scan (settings: λ_(EX)=495 nm, S_(EX)=3.0 nmS_(EM)=7.0 nm) was carried out on dilute labelled RNA (1:100 1× bindingbuffer), a control (1 pmol Fluorescein-12-UTP) was added and the changein fluorescence observed. Subsequently, T3 was added to labelled RNA inratios of 1:2, 2:2, 4:2 and 6:2 and changes in fluorescence signals wereobserved.

Effect of T3 RNA Aptamer (SEQ ID NO:38) on the Fibril Formation Assay

In order to determine the effect of the T3 RNA aptamer (SEQ D NO: 38) onfibril growth assay, 600 μLs 50 μM fibril growth assay as hereinbeforedescribed was prepared. The following reagents and apparatus were used:10 mm×10 mm×4 mm Styrofoam (to provide increased height of cuvette);˜440 μg purified unlabelled transcript of T3 clone (SEQ ID NO:38);dried, purified ‘Mixed’ Aβ(1-40); 1× binding buffer, magnetic stirrer;Fluorimeter LS 50B and; Formvar coated copper grids, 4% (w/v) uranylacetate.

Fluorescence signal was observed and when the signal started to increaseslowly after 30 minutes 20 μLs water was added and the effect observed.After a further 2-3 minutes the signal returned to baseline and 20 μLsT3.2 RNA (˜440 μgs) (SEQ ID NO: 38) was added and the effect observed.The experiment was repeated using ⅕^(th) amount T3 RNA (SEQ D NO: 38)(˜88 20 μs).

β2-Microglobulin Peptide

β_(2m) Preparation & Fibril Formation

The β2m protein was over-expressed, extracted and purified as previouslydescribed¹⁶. Purification was carried out using an ÄKTA Explorer(Amersham Pharmacia Biotech) with a HiTrap Mono Q ion exchange column.

Immature fibril formation was carried out using a composite bufferconsisting of 25 mM TRIS, 25 mM MES, 25 mM glycine, 25 mM sodium acetate(TMGA buffer). HCl was added to adjust to pH3.6 before the addition ofsodium chloride to bring the final ionic strength to 400 mM. β₂M wasadded to a final concentration of 1 mg ml⁻¹ and the mixture incubated at37° C. for 72 hrs. Unfolding and fibrillogenesis in β₂M can be triggeredby altering pH and ionic strength.

Mature fibril formation was carried out using a composite bufferconsisting of 25 mM sodium acetate & 25 mM sodium phosphate. HCl wasused to adjust the pH to 2.5. No additional salt was included. β₂M wasadded to a final concentration of 2 mg ml⁻¹ and incubated at 37° C. on ashaker 150 rpm for 7 days.

Fibrils were characterised by Thioflavin-T binding assay and electronmicroscopy. In both types of fibril formation reaction, a 10 μsample isremoved and added to 990 μl 10 μM Thio-T buffered with 10 mM Tris HClpH8.5. The average fluorescence emission signal over a 30 second scan isthen measured using a Perkin-Elmer Luminescence Spectrometer (LS50 B)set at 444 nm (excitation) and 480 nm (emission). The presence ofimmature fibrils is indicated by a ˜10 fold increase in emission overthat of Thio-T alone. Mature fibrils typically give a ˜20 fold increaseover that of Thio-T alone. Addition of native state β₂M gives a marginalincrease in fluorescence (FIG. 8),

Fibrils of both types can be viewed using electron microscopy.Copper-coated EM grids are placed coated side down onto a 30 μl drop ofbuffered fibrils for 30 seconds. Excess fibrils are removed by dotingthe grid onto a 30 μl drop of double distilled water, before placing thegrid onto 4% (w/v) uranyl acetate for 45 seconds. Excess stain isremoved by dotting onto a fresh drop of double distilled water. The gridis then allowed to air dry before viewing using a JEOL 1200 electronmicroscope.

Magnetic Bead Derivatisation

Native and fibrillar β₂M were biotin tagged using the ‘EZLink’(Sulfo-NHS-LC-LC-biotin) reagent from PIERCE. The protocol supplied withthe reagent had to be modified as the biotinylation reaction takes placeat neutral pH (β₂M fibrils rapidly dissociate above pH5). ‘EZLink’ isdissolved in DMSO to a final concentration of 1 mg ml⁻¹. 30 μl of 2MTris pH 10.8 is added to 500 μl of preformed fibrils (˜1 mg ml⁻¹) toraise the pH above 6. 75 μl of EZLink is added, mixed briefly by gentlepipetting and incubated on ice for 15 mins. The pH is reduced and excessNHS biotin is removed by placing the entire reaction into a ‘Slidalyzer’cassette (3500 Mw cut off) and dialysing against 1 L fibril formationbuffer for 2 hours. The dialysis buffer is changed after 1 hour.Streptavadin coated beads, purchased from Dynal, were derivatised withnative β₂M, immature fibrils or mature fibrils as described in theDynabeads protocol.

SELEX

All rounds of in vitro selection were carried out on a Biomek 2000automated workstation (Beckman Coulter) using methods adapted from thosedescribed by Cox et al, ¹⁸. The Biomek has an integrated PTC-200thermocycler with heated power bonnet (MJ Research), a multiscreenfiltration system & vacuum manifold (Beckman Coulter) and a ThermalExchange Unit (Beckman Coulter) with a Thermal 48 cooling block(Acme-Automation). The Biomek and all integrated components arecontrolled using Bioworks 3.1c (Beckman Coulter).

To produce the initial DNA pool; 20 μl 10×PCR buffer, 1.6 μl 10 mMdNTP's, 10 μl 10 μM sense primer JAN01P1 (P₁) (SEQ ID NO:108), 10 μl 10μM anti-sense primer JAN01P2(P₂) (SEQ ID NO:109), 20 μl 0.1 μM templateDNA JAN01T (SEQ ID NO:110), 6 μl 50 mM MgCl₂, 2 μl Taq DNA polymeraseand 124.4 μl R⁻H₂O are thoroughly mixed and divided into 50 μl aliquots.These are then placed into a Techne Progene thermocycler and cycled asfollow; 94° C. for 90 sec (Hot start), 8 cycles of: 94° C. for 45 sec(Denature); 50° C. for 60 sec (Anneal) and 72° C. for 90 sec (Extend)

As this model has a heated lid to prevent evaporation, the addition ofmineral oil is not necessary The efficiency of PCR was assessed bypolyacrylamide gel electrophoresis (PAGE).

RNA Pool

Transcription reactions were carried out by adding 5 μl 10×transcription buffer (to give final concentrations of 40 mM Tris-HClpH7.9, 26 mM MgCl₂, 2.5 mM spermidine, 5 mM DTT, 0.01% Triton X-100), 16μl R⁻H₂O, 16 μl 25 mM NTP mix, 10 μl template DNA. 40U RNasin and 100 UT7 RNA polymerase were then added to give a final volume of 50 μl. Aftermixing by rapid aspiration and dispensing, the transcription mix wasincubated at 37° C. for 90 mins in the thermocycler.

Selection

20 μl derivatised Dynabeads (approximately 6.6×10⁶ beads) are added to80 μl binding buffer and mixed by rapid aspiration and dispensing. Thebinding buffer used for the selections is the same TMGA buffer used forfibril formation. In the native β₂M selections, TMGA buffer pH7 is used.The entire transcription reaction is mixed into this. Mixing is repeatedafter a 5 min. room temperature incubation, to ensure the beads do notsettle out. Partitioning of bound from unbound species is carried out byfiltration through a multiscreen 96-well plate (PVDF membrane) suppliedby Millipore. The Dynabeads are then rinsed by resuspending in bindingbuffer and repeating the filtration. Bound RNA species are eluted byresuspending the Dynabeads in 53 μl R⁻H₂O and incubating at 95° C. for15 mins.

RT-PCR Amplification

The following are added to the eluted RNA; 5 μl 10 mM primer 1, 5 μl 10mM primer 2, 2 μl 10 mM dNTPs and 33 μl RT-PCR buffer to give finalconcentrations of 10 mM Tris HCl pH8.4, 50 mM KCl, 5% acetamide, 0.05%Nonidet P40. This is heated to 65° C. for 10 mins. After reducing thetemperature to 50° C., 200 U Superscript II™ reverse transcriptase and5U Taq DNA polymerase are added. This is thermocycled as follows: 50° C.for 30 mins (Reverse transcribe), 8 cycles of: 94° C. for 45 see(Denature); 50° C. for 60 sec (Anneal) and72° C. for 90 sec (Extend).

10 μl of the RT-PCR product was used as a template in the transcriptionreaction for the next round. After 10 successful rounds of SELEX,samples of the RT-PCR products are cloned and sequenced.

Measuring Aptamer Binding Affinity by Surface Plasmon Resonance

Individual RNA aptamers are produced by PCR amplification and gelpurification of the aptamer sequence from an individual clone. This isthen used as the template DNA in a 50 μl transcription reaction (ascarried out in vitro selections). Transcripts are DNase 1 treated toremove the template before phenol-chloroform extraction and ethanolprecipitation.

All SPR experiments are carried out using a BIAcore 2000. TMGA bufferpH3.6 was used as running buffer. An SA chip (Dextran matrixpre-immobilised with streptavidin) was washed by carrying out threeinjections of 50 μl ‘chip preparation’ solution (50 mM NaOH, 1 M NaCl)at 50 μl min⁻¹ to remove any loosely bound streptavidin. Each flow-cellwas derivatised with a different form of β₂M by injecting 150 μl ˜50 μMβ₂M (Native, immature or mature fibrils) across a single flow-cell at 30μl min⁻¹. Excess β₂M was removed by washing the flow-cells through witha 150 μl injection of running buffer.

EXAMPLE 1

Aptamers N2 and F2 (SEQ ID NOS 61 & 74) were passed across eachflow-cell (30 μl, 0.5 μM RNA at 10 μl min⁻¹). A selection ofanti-native-β_(2m) aptamers were passed across the blank and nativeβ_(2m) flow-cells at concentrations of 1 μM, 0.5 μM & 0.1 μM. TheBIAevaluation software was used to correct the sensorgrams for ResonanceUnit (RU) changes that are due to differences in the buffers rather thanactual interactions. The software was then used to determine the Kd foreach aptamer based on the corrected sensorgrams. Aptamer Kd at 1 μM Kdat 0.5 μM Kd at 0.1 μM N2 (SEQ ID NO: 61) 1.09 μM 1.91 μM 89.1 nM N4(SEQ ID NO: 63). 104 nM 317 nM N5 (SEQ ID NO: 64). 785 nM 300 nM 845 nMN7 (SEQ ID NO: 66). 2.58 μM 379 nM N8 (SEQ ID NO: 67). 382 nM 343 nM73.2 nM N11 (SEQ ID NO: 70). 505 nM 329 nM 35 μM N13 (SEQ ID NO: 71).332 nM 1.46 μM 49.2 nM

EXAMPLE 2

Our aptamers against the Aβ1-40 and β2m species (SEQ ID NOS: 1-55 and58-71 and SEQ ID NOS: 72-90) have been modified with fluorescent labelsby simple inclusion of fluorescein-labelled UTP in in vitrotranscription reactions. With reference to FIG. 2 differential quenchingand wavelength shifting in the presence of differing forms of the β2mtarget is shown with anti-fibril β2m aptamer (F2) (SEQ ID NO 74),labelled with fluorescein-UTP. The fluorescence properties of thesemolecules are sensitive to their bound state and may be the basis ofsimple diagnostic screening and imaging reagents. For instance, thestate of disease progression may be judged by staining/screening withdifferently labelled aptamers directed against monomer, pre-fibril orfibrillar species.

EXAMPLE 3

Anti-β2m aptamers have been isolated against native, monomeric proteinas well as various amyloid fibrillar forms. Suitably modified versionsof our aptamers against the non-fibrillar forms of β2m may be added tothe blood during dialysis, stabilising that species with respect tofibril formation. Post-dialysis the aptamers will slowly hydrolyse inthe body to harmless natural bi-products. Our anti-amyloid aptamersdirected against β2m, were selected at each round aftercounter-selection against the native monomer. Remarkably, this approachhas advantageously yielded reagents that bind to amyloid fibrils verytightly, dissociating very slowly, and in addition advantageously havelittle or no affinity for the native protein. Clearly, these reagentscan be used directly to monitor early stages of amyloidosis in patientsunder-going dialysis. Since the anti-native aptamers do cross-react withthe amyloid forms of the protein, these results show for the first timethat apatopes of the native β2m are still present in the amyloid fibre.In addition, the fibril specific species appear to be recognisingamyloid-specific apatopes, suggesting that these may be common to allamyloid fibrils, massively extending their utility.

With reference to FIG. 3A we have shown by surface plasmon resonancebinding curves (sensorgrams) of the anti-F2 β2m aptamer (SEQ ID NO 74)binding to: a) an underivatised flow cell (red, that the signal fallsdue to differences in refractive index of the sample); b) a flow cellderivatised with native β2m (magenta, that binding is detectable underthese conditions); c) a flow cell derivatised with β2m fibrils (green,that there is strong binding with slow dissociation) and d) a flow cellderivatised with mature β2m fibrils (blue, result as in (c)).

With reference to FIG. 3 B, we have shown by a similar method thebinding of an anti-native aptamer (SEQ ID NO 61) to the same immobilisedtargets using SPR. The sensorgram with native β2m as target (magenta)shows binding with rapid dissociation kinetics. This aptamer also bindsstrongly to the fibrillar targets suggesting that the apatopes beingrecognised is conserved after fibril formation. In the case of thefibrils the RU response is larger because there is more proteinimmobilised in these cases. Also, the dissociation behaviour is clearlydistinct from that seen with the native monomer, being significantlyslower. This is consistent with dissociation and rebinding because ofthe macromolecular nature of the fibrillar target.

EXAMPLE 4

We have selected aptamer sequences against the D-amino acid forms ofAβ1-40. The self-aggregation behaviour of these non-natural Aβ1-40peptides has been carefully assessed and three selection targetsidentified (FIG. 4). These are 1) T1 the monomeric peptide; 2) T2 thepre-fibrillar state and 3) T3 the protofibrils that are the directprecursors of mature filaments. This work therefore is a significantadvance over the previous report of anti-Alzheimer plaques in whichnatural peptides were used and their state of aggregation was notassessed. Sequence comparisons of the aptamers isolated against eachtarget suggest that there are amyloid specific apatopes in T2 and T3that are not present in T1. The amyloid specific aptamers have someremarkable properties that make them potentially useful compounds. Forexample, one anti-T3 aptamer (SEQ ID NO: 38) binds its cognate targetco-operatively (FIG. 5B), when labelled with fluorescence it showsspecific fluorescence quenching upon amyloid binding, and therefore is apotential diagnostic and/or screening agent. It also dramaticallyreduces fibril formation when added to an in vitro fibrillation assay(FIG. 6).

EXAMPLE 5

Experimental results with biotinylated β2m have shown that β₂m fibrilsmay be biotinylated with more than one biotin molecule. With referenceto FIG. 7, peak A shows unbiotinylated β2m fibrils, the 452 Da gap topeak B is indicative of a single biotin molecule and the further 452 gapto peak C that the fibril carries a second biotin molecule. Similarly,the next 452 gap to peak D illustrates that the fibril can be associatedwith a third biotin molecule. Results have shown (FIGS. 8 and 9) thatbiotinylation of both immature and mature β2m fibrils allows the targetto be successfully attached to a bead and that biotinylation does notaffect the integrity or substantially alter the properties of fibrilscompared to underivatised fibrils.

EXAMPLE 6

Studies on β2m fibril formation with two randomly selected aptamers F3.2and NR10 (corresponding to SEQ ID NO 74 and the pool of aptamersgenerated after the 10^(th) selection round, respectively) have shownprofound effects on fibril formation over time (FIG. 10). Theanti-fibril aptamer, F3.2, shows distinct inhibitory properties withrespect to fibril formation. Strikingly, the NR10 pool appears to allowonly an initial burst of fibril formation followed by disassembly of thelight-scattering species being generated, i.e. it appears capable ofcompletely ablating fibril formation. Both aptamers were found tosignificantly reduce fibril formation compared to the control reactionof β2m with the naïve pool. Interestingly, β2m fibril formation appearsto be enhanced in the control sample, which may indicate that there arespecies in the starting RNA pool that can actively promote fibrilformation. This may provide a unique insight into the natural mechanismof amyloid formation in vivo.

EXAMPLE 7

Structure of human β₂m is shown in FIG. 11 and corresponding the aminoacid sequence is shown in FIG. 12, and is coloured from the N-terminusof the protein (red) to the C-terminus (violet). The location ofelements of secondary structure was determined using DSSP.Individual-strands are labelled A though G. The disulphide bond thatlinks Cys 25 (strand B) and Cys 80 (strand F) is also shown. The figurewas made using the co-ordinates 1DUZ.PDB using the programme MOLSCRIPTand RASTER 3D. (b) The amino acid sequences of the peptides of β₂mstudied here. With the exception of Ilel and Met99, all peptidesequences were acetylated at their N-termini and amidated at theirC-termini.

Results have shown, only two sequences, both of which encompass theregion that forms strand E in native β₂m, are capable of formingamyloid-like ffbrils in vitro. These peptides correspond to residues59-71 (peptide E) and 59-79 (peptide E′) of intact₂m. The peptides formfibrils under the acidic conditions shown previously to promote amyloidformation from the intact protein (pH<3 at low ionic strength and pH<5at high ionic strength), and also associate to form fibrils at neutralpH. Fibrils formed from these two peptides enhance fibrillogenesis ofthe intact protein.

Accordingly, we believe that residues 59-79 are important in theself-association of partially folded ₂m into amyloid fibrils and arepotentially involved in the assembly mechanism of the intact protein invitro. Thus, these residues represent a target against which aptamersmay be directed and these residues provide a hitherto unrecognisedtarget site for developing amyloid disease therapeutics.

REFERENCES

-   1. Sunde, M. & Blake, C. C. (1998). From the globular to the fibrous    state: protein structure and structural conversion in amyloid    formation Q Rev Biophys 31, 1-39.-   2. Fink, A. L. (1998). Protein aggregation: folding aggregates,    inclusion bodies and amyloid Fold Des. 3, R9-R23.-   3. Kelly, J. W. (1998). The alternative conformations of    amyloidogenic proteins and their multi-step assembly pathways Curr.    Opin. Struct. Biol. 8, 101-106.-   4. Selkoe (1994) Normal and abnormal biology of the beta-amyloid    precursor protein Ann. Rev. Neurosci., 17, 489-517;-   5. Rochet et al. (2000). inhibition of fibrillization and    accumulation of prefibrillar oligomers in mixtures of human and    mouse alpha-synuclein Biochem. 39, 10619-10626.-   6. Bucciantini et al. (2002). Inherent toxicity of aggregates    implies a common mechanism for protein misfolding diseases Nature    416, 507-511.-   7. Walsh et al. (2002). Amyloid-beta oligomers: their production,    toxicity and therapeutic inhibition Biochem Soc Trans 30, 552-557.-   8. Dobson, C. M. (1999). Protein misfolding, evolution and disease    Trends in Biochemical Sciences 24, 329-332.-   9. Check (2002) Nerve inflammation halts trial for Alzheimer's drug    Nature, 415, 462.-   10. Gejyo et al. (1986). Serum levels of beta 2-microglobulin as a    new form of amyloid protein in patients undergoing long-term    hemodialysis New Eng. J. Med 314, 585-586.-   11. Gejyo et al. (1985). A new form of amyloid protein associated    with chronic hemodialysis was identified as beta 2-microglobulin    Biochem Biophys Res Commun 129, 701-706.-   12. James W, Encyclopaedia of Analytical Chemistry. R. A Mayers (Ed)    4848-4871.-   13. Rusconi et al. (2002) RNA aptamers as reversible antagonists of    coagulation factor IXa Nature, 419, 90-94;-   14. Ylera et al. (2002) Selection of RNA aptamers to the Alzheimer's    disease amyloid peptide BBRC, 290, 1583-1588;-   15. Jones et al. Amyloid-forming peptides from    β2-microglobulin—Insights into the mechanism of fibril formation in    vitro. J. Mol. Biol. in press.-   16. Yamaguchi et al. (2001). Apolipoprotein E inhibits the    depolymerization of β2-microglobulin-related amyloid fibrils at a    neutral pH. Biochemistry, 40, 8499-8507.-   17. McParland et al. (2000). Partially unfolded states of beta    2-microglobulin and amyloid formation in vitro Biochem. 280,    5678-5699.-   18. Kad et al. (2001). Beta(2)-microglobulin and its deamidated    variant, N17D form amyloid fibrils with a range of morphologies in    vitro J Mol Biol 313, 559-571.-   19. Cox et al (1998) Automated RNA selection Biotech. Prog., 14,    845-850

1. A purified and isolated non-naturally occurring nucleic acid ligandto a fibrillar protein target, wherein said ligand is an RNA ligandselected from the group consisting of: (i) the nucleic acid depicted inany one of SEQ ID NOS: 1-55 or 58-105; (ii) having the corresponding DNAor RNA sequences of any one of SEQ ID NOS: 1-55 or 58-105 or thecorresponding fully complementary sequences thereof or their L-ribosederivatives; and (iii) derivatives of the sequence depicted in any oneof SEQ ID NOS: 1-55 or 58-105 having at least about 60%, 70%, 80% or 90%sequence identity to any one of the nucleotide sequences, and which havea binding affinity to a fibrillar protein.
 2. The nucleic acid ligandaccording to claim 1 which is substantially homologous to and hassubstantially the same ability to bind said fibrillar protein target asa ligand comprising the nucleic acids depicted in any one of SEQ ID NOS:1-55 or 58-105.
 3. The nucleic acid ligand according to claim 1, whereinthe nucleic acid has substantially the same structure and the sameability to bind said fibrillar protein target as a ligand selected fromthe group comprising the nucleic acids depicted in any one of SEQ IDNOS: 1-55 or 58-105.
 4. The nucleic acid according to claim 1 whereinthe fibrillar protein target is selected from the group consisting ofmonomeric β2m or Aβ1-40 or Aβ1-42, protofibrillar β2m or Aβ1-40 orAβ1-42 and mature fibrillar β2m or Aβ1-40 or Aβ1-42.
 5. The nucleic acidaccording to claim 1 wherein the fibrillar protein target compriseseither L- or D-amino acid molecules.
 6. The nucleic acid according toclaim 1 wherein the nucleic acid of any one of SEQ ID NOS: 1 to 16 has abinding affinity to a D-amino acid Aβ1-40 monomeric target.
 7. Thenucleic acid according to claim 1 wherein the nucleic acid of any one ofSEQ ID NOS: 17 to 36 has a binding affinity to a D-amino acid Aβ1-40pre-fibrillar target.
 8. The nucleic acid according to claim 1 whereinthe nucleic acid of any one of SEQ ID NOS: 37 to 55 has a bindingaffinity to a D-amino acid Aβ1-40 protofibril target.
 9. The nucleicacid according to claim 1 wherein the nucleic acid of any one SEQ IDNOS: 58 to 71 has a binding affinity to a native β2-microglobulinprotein target.
 10. The nucleic acid according to claim 1 wherein thenucleic acid of any one of SEQ ID NOS: 72 to 90 has a binding affinityto a β2-microglobulin immature fibril protein target.
 11. The nucleicacid according claim 1 wherein the nucleic acid of any one of SEQ IDNOS: 91 to 105 has a binding affinity to a β2-microglobulin maturefibrillar protein target.
 12. The nucleic acid according to claim 1further comprising any one or more of the following features: (i) afluorescent label; (ii) an imaging label; and (iii) a flanking region.13. The nucleic acid according to claim 12 wherein the flanking regioncomprises any one or more nucleic acid sequences selected from the groupconsisting of SEQ ID NOS: 56, 57, 106 and
 107. 14. A vector comprisingat least one or more nucleic acids of claim
 1. 15. A host cellcomprising at least one or more nucleic acids of claim 1 or a vectorcomprising the at least one or more nucleic acids of claim
 1. 16. Use ofa binding motif comprising a peptide sequence derived from human β2mthat retains the ability of the whole protein to form amyloid fibrils,as a target for selecting a nucleic acid ligand.
 17. Use of a peptidesequence comprising any one of SEQ ID NO: 111, 112 or 113 or derivativesor variants thereof that retain the ability of the whole protein to formamyloid fibrils, as a target for selecting a nucleic acid ligand.
 18. Apurified and isolated non-naturally occurring nucleic acid ligand to afibrillar protein target, wherein the target comprises the binding motifof claim
 16. 19. A purified and isolated non-naturally occurring nucleicacid ligand to a fibril cross β-core protein target.
 20. Apharmaceutical composition comprising at least one nucleic acid of claim1 or the vector comprising the at least one or more nucleic acids ofclaim
 1. 21. A pharmaceutical composition according to claim 20comprising at least two nucleic acid ligands each with bindingaffinities for the same or different forms of a fibrillar protein.
 22. Apharmaceutical according to claim 20 further comprising a suitableexcipient, diluent or carrier.
 23. Use of a nucleic acid according toclaim 1 for the manufacture of a medicament for treating amyloiddiseases.
 24. Use according to claim 23 for the treatment of Alzheimer'sand DRA disease conditions.
 25. A method of treating a patient sufferingfrom Alzheimer's disease or a disease associated with amyloid formationcomprising administering a therapeutically effective amount of (i) thenucleic acid ligand according to claim
 1. (ii) a vector comprising thenucleic acids of claim 1, or (iii) a pharmaceutical compositioncomprising (i) or (ii).
 26. A method according to claim 25 wherein thetherapeutically effective amount of a nucleic acid ligand, vector orpharmaceutical composition is administered by an intra-venous,intra-muscular, intra-peritoneal route and optionally is administered onmore than one occasion.
 27. Use of the nucleic acid according to claim 1or a vector comprising the nucleic acids of claim 1 as a diagnosticagent for detecting the presence and/or progression of an amyloiddisease.
 28. A method of monitoring the presence and/or progression ofan amyloid disease comprising: (a) administering to a patient (i) thenucleic acid ligand according to claim 1, (ii) a vector comprising thenucleic acids of claim 1, or (iii) a pharmaceutical compositioncomprising (i) or (ii); (b) imaging the presence of binding of saidnucleic acid ligand to an amyloid fibril; and (c) optionally repeatingthe process at a later date to assess presence or progression of adisease state.
 29. A method for the isolation of nucleic acid ligands toa fibrillar protein target comprising: (i) preparing a candidate mixtureof nucleic acids; (ii) contacting the candidate mixture of nucleic acidswith a biotinylated immobilised fibrillar protein on ice, whereinnucleic acids having an increased affinity to the fibrillar proteinrelative to the candidate mixture are partitioned from the remainder ofthe candidate mixture; (iii) partitioning the increased-affinity nucleicacids from the remainder of the candidate mixture; (iv) amplifying theincreased-affinity nucleic acids to yield a mixture of nucleic acidswith relatively high affinity and specificity for binding to thefibrillar protein, whereby a nucleic acid ligand of the fibrillarprotein may be identified.
 30. A method according to claim 29 whereinthe candidate mixture comprises single stranded nucleic acids.
 31. Amethod according to claim 30 wherein the single stranded nucleic acidscomprise ribonucleic acids.
 32. The method according to claim 29 furthercomprising modifying the nucleic acid ligand with a fluorescent labeland/or an imaging reagent.
 33. A method according to claim 29 whereinthe nucleic acid ligand further comprises a flanking selected from thegroup consisting of SEQ ID NO: 56, 57, 107 and
 108. 34. A nucleic acidproduct identified and isolated according to the method of claim 29.